The development of transposon vectors for mammalian transgenesis has the potential to solve many of the limitations of retroviral (e.g., Lentiviral) vectors such as limited transgene size and random integration. We have recently demonstrated that the piggyBac transposon, originally isolated from the cabbage looper moth Trichoplusia ni, is more efficient at transposition than hyperactive Sleeping Beauty, once thought to be the most active transposon. Our first aim explores the possibility of a single plasmid transposase-transposons (Helper and Donor) ability to enzymatically insert a transgene in the transposon into the hosts genomic chromosome, at the teranucleotide (TTAA) site. We intend to use the information gained during cell transfection for the production of transgenic mice. Several methods have been developed for producing transgenic mice, including pronuclear microinjection, ICSI-Tr, virus-mediated insertion, and ES cell-mediated approaches. Of these only the virus-mediated insertion (Lentiviral) employs an active mode of transgene insertion, with the others relying on the repair mechanism of the oocyte for transgene integration. Since the development of ICSI-Tr which works optimally only with freeze-thawed sperm, we concentrated on improving the integration of transgenes in mice by developing active transgenesis procedures. Among approaches with protein recombinases and transposases, the hyperactive Tn5 transposase protein (*Tn5p) was by far the most efficient method when introducing the transgene in a transposon along with non freeze-thawed spermatozoa into unfertilized oocytes (TN:ICSI). However, because TN:ICSI suffers from cumbersome enzyme preparation techniques, we have now moved away from the enzymatic insertions of transgenes and developed DNA based procedures that allow synthesis of the transposase in-situ. Furthermore, to achieve target specificity, we fused the GAL4 DNA binding domain to the N-terminal of the piggyBac protein and determined the activity of the chimeras by chromosome integration assays. The GAL4-piggyBac transposase displayed an activity similar to that of wild-type piggyBac and inserted at its regular TTAA site. We therefore suggest that this transposon system, because of its flexibility for molecular engineering and its relatively high transposition activity, could be ideal for mammalian transgenesis and pre-clinical gene therapy experiments. For the first specific aim, we will additionally utilize a chimeric transposase coupled to the GAL-4 DNA binding domain for targeted integration into a mouse embryos genome with a UAS tandem array. In the second aim, we will develop transposases coupled to a zinc finger DNA binding domain, both in DNA and cRNA form, that recognize the tyrosinase locus for targeted genomic integration in both cell lines and animals. Therefore, this grant should lead to improved non-viral integrating vectors for use in mammalian transgenesis and animal gene therapy experiments. [unreadable] [unreadable] [unreadable] [unreadable]